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The objective of this observational experiment was to determine the association between the interval from reaching an activity threshold (AT) using an automated activity monitoring system (AAM) and the time of artificial insemination on pregnancy per AI (P/AI) for lactating Holstein cows inseminated with either fresh or frozen semen. Lactating Holstein cows (n = 5,200) from 8 commercial dairy farms were inseminated based on visual heat detection and/or based on the alert of an accelerometer system (Heatime; SCR Engineers Ltd., Netanya, Israel). Data were analysed using the GENLINMIXED procedure in SPSS. Onset of estrus was defined as the time when the AT (index value ≥ 35) was reached. The mean (± standard deviation) interval from onset of estrus to AI was 13.8 ± 8.9 h. Pregnancy per AI was affected by parity (p = 0.01), THI one week before AI (p = 0.01), estrus intensity (p = 0.01), AI number (p = 0.01), milk yield one week before AI (p = 0.03), type of semen (p = 0.05), and the interval from reaching AT to AI (p = 0.01). The interaction of type of semen with interval from reaching AT to AI (p = 0.47) had no effect on P/AI. There was a quadratic effect of the interval from reaching AT and AI on probabilities of pregnancy. Pregnancy per AI at 35 d after AI was highest for cows inseminated between 7 to 24 h after onset of estrus (‐24 to 0 h = 32.0%, 1–6 h = 32.6%, 7–12 h = 42.2%, 13–18 h = 45.0%, 19–24 h = 43.7%, 25–48 h = 36.0%). In conclusion, inseminating cows 7 to 24 h after passing the AT yielded the highest P/AI irrespective of type of semen. Inseminating cows before or 6 h after reaching AT was detrimental on P/AI. Supported by FBF.